Process for increasing ionic charge in mass spectrometry

ABSTRACT

Processes and apparatus are described for the analysis of molecules or fragments thereof, which are capable of carrying multiple charges, by reacting the multiply charged molecules or fragments thereof with other ions using mass spectrometry.

FIELD OF THE INVENTION

This invention relates to the analysis of molecules by massspectrometry. In particular, this invention relates to the analysis ofmolecules that may possess multiple charges by mass spectrometry byincreasing the charge on the molecules.

BACKGROUND OF THE INVENTION

The analysis of large molecules by mass spectrometry is playing anincreasingly important role in modern biological research. Such analysisis facilitated by recent advances in the ability to generate gas-phaseions of these large molecules. Mass spectrometry is particularly suitedto the analysis of these biological materials because they are oftenavailable only in small quantities, typically as isolates from naturalsources. In addition, mass spectrometry is well suited to the area ofproteomics, which includes the study of the time-dependent proteincomplement of an organism.

Large biologically derived molecules are often biopolymers such asproteins, peptides, nucleic acids, oligonucleotides, polysaccharides,and oligosaccharides. The identification and analysis of thesebiopolymers are dependent on a sufficient amount of fragments, and asufficient diversity in the fragmentation patterns that may be generatedduring mass spectrometry. These fragments and fragmentation patterns areinterpreted to derive the primary sequence of the biopolymers. Further,the number of and diversity of the fragments generated by massspectrometry is often dependent upon the number of and diversity of thecharges and charge states that may be generated on the molecule.Processes and apparatus for manipulating and modifying these chargestates to optimize the observed fragmentation will expand the field ofmass spectrometric analysis of biologically derived molecules.

SUMMARY OF THE INVENTION

Processes for analyzing molecules by mass spectrometry are describedherein. These processes use the technique of generating an initialcharge on the molecule to be analyzed, placing that molecule into agaseous phase, and manipulating or modifying that initial charge.

The initial charge is generated using techniques that include but arenot limited to plasma desorption ionization (PDI), field desorptionionization (FDI), electrohydrodynamic ionization (EHI), laser desorptionionization (LDI), matrix assisted laser desorption ionization (MALDI),atmospheric pressure matrix assisted laser desorption ionization(APMALDI), spray ionization techniques, including electrosprayionization (ESI), nano-electrospray ionization, thermospray ionization(TSI), aerospray ionization (ASI), sonic spray ionization (SSI),particle desorption methods such as fast atom bombardment (FAB), fastion bombardment (FIB), such as secondary ion mass spectrometry (SIMS),and massive cluster impact, and the like.

After the initial charge is generated, an ion storage or trappingprocedure is performed. Ions may be stored or trapped in any of avariety of devices including but not limited to linear ion traps,quadrupole ion traps, and the like. In some embodiments of the processesdescribed herein, the initially generated ions are directed to the ionstorage device by illustratively using a turning quadrupole, and thelike.

In one illustrative embodiment, the initial charge is manipulated bycontacting the initially charged molecule, or any charged fragments ofthat molecule, with at least one multiply charged ionic compound. Theinitial charge may be a single charge of either polarity or a multiplecharge of either polarity. After contacting the initially chargedmolecule or charged fragment thereof with at least one multiply chargedionic compound, the initial charge on the molecule or fragment thereofis changed. Illustratively, the absolute value of the initial charge islowered, or the polarity of the initial charge is changed. In the lattercase where the polarity of the initial charge is changed, the absolutevalue of the resulting charge may therefore be greater than the absolutevalue of the initial charge.

In another illustrative embodiment, the initially charged molecule orcharged fragment thereof is contacted with more than one multiplycharged ionic compound. After contacting the initially charged moleculeor charged fragment thereof with one multiply charged ionic compound,the initial charge on the molecule or fragment thereof is changed.Illustratively, the absolute value of the initial charge is lowered, orthe polarity of the initial charge is changed. In the latter case wherethe polarity of the initial charge is changed, the absolute value of theresulting second charge may therefore be greater than the absolute valueof the initial charge. Subsequently, the molecule or fragment thereofhaving the resulting second charge is contacted with a second multiplycharged ionic compound. After contacting the molecule or fragmentthereof having the second charge with the second multiply charged ioniccompound, the resulting second charge on the molecule or fragmentthereof is changed. Illustratively, the absolute value of the secondcharge is lowered, or the polarity of the second charge is changed. Inthe latter case where the polarity of the second charge is changed, theabsolute value of the resulting third charge may therefore be greaterthan the absolute value of the second charge.

In one aspect, the polarity of initial charge is changed, and theresulting charge has an absolute value greater than the absolute valueof the initial charge. In another aspect, the initial charge has anabsolute value greater than one, and the resulting charge has anabsolute value of one and the opposite polarity of the initial charge.

In another aspect, the polarity of the initial charge is changed aftercontacting a multiply charged ionic compound, and the polarity of theresulting second charge is changed back, or reverted, to the polarity ofthe first charge after contacting a second multiply charged ioniccompound. In another aspect, the absolute value of the final charge isgreater than the absolute value of the initial charge.

Prior to the manipulation or modification of the charge on the moleculeor fragment thereof, or following the manipulation or modification ofthe charge on the molecule or fragment thereof, an optional massfiltering step is performed on one or more of the ions generated in theprocesses described herein. This mass selection step may be performed onthe molecular ions, including fragment ions thereof prior to contact, orfollowing contact with the multiply charged ionic compounds. This massselection step may also be performed on any of the multiply chargedionic compounds that are contacted with the molecular ions or fragmentions thereof.

Prior to the manipulation or modification of the charge on the moleculeor fragment thereof, or following the manipulation or modification ofthe charge on the molecule or fragment thereof, an optional massanalysis step is performed on one or more of the ions generated in theprocesses described herein. The mass analysis step may use any of avariety of techniques including but not limited to scanning from alinear ion trap, scanning from a quadrupole ion trap, time of flight(TOF), ion cyclotron resonance, and the like.

The processes described herein may also include a step of concentratingthe charge of the molecule or fragment thereof using any of a variety oftechniques including but not limited to atmospheric sampling glowdischarge ionization (ASGDI), and the like.

Apparatus for performing the processes described herein are alsodescribed. Various configurations are constructed from those componentsnecessary to perform the steps of the processes described herein,including (1) various sources for generating ions or charges onmolecules or analytes to be analyzed, (2) various components for storingor trapping those molecular ions or fragment ions thereof so-generated,including components for directing ions to such storage components, (3)various components for optionally filtering or selecting certain massesfrom those molecular ions or fragment ions thereof, (4) variouscomponents for providing a reaction environment for contacting othercharged or multiply charged ionic compounds with those molecular ions orfragment ions thereof to modify the charge states of the molecular ionsor fragment ions, and (5) various components for analyzing the molecularions or fragment ions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an apparatus 10 for performing processes describedherein.

FIG. 2A illustrates an ESI mass spectrum of bradykinin showing the[B+H]⁺ ion. The vertical axis is relative abundance in arbitrary units.

FIG. 2B illustrates a mass spectrum of bradykinin following treatment ofthe monocation of bradyknin with a dianion of benzene-1,4-bispropionicacid (PDPA) showing the [B−H]⁻ ion. The vertical axis is relativeabundance in arbitrary units.

FIG. 2C illustrates a mass spectrum of bradykinin following treatment ofthe monoanion of bradykinin with a hexacation ofpoly(propyleneimine)dendrimer showing the [B+H]⁺ ion and the [B+2H]²⁺ion. The vertical axis is relative abundance in arbitrary units.

FIG. 3A illustrates an ESI mass spectrum of bradykinin showing the[B+H]⁺ ion. The vertical axis is relative abundance in arbitrary units.

FIG. 3B illustrates an ESI mass spectrum of carboxylate terminatedpolyamidoamine dendrimer (generation 0.5) (PAMAM) showing multiplecharge states on the dendrimer. The vertical axis is relative abundancein arbitrary units.

FIG. 3C illustrates a mass spectrum of bradykinin following treatment ofthe monoanion of bradykinin with a hexacation of poly(propylenimine)(1,4-diaminobutane (DAB) core, generation 4) dendrimer showing the[B+H]⁺ ion and the [B+2H]²⁺ ion. The vertical axis is relative abundancein arbitrary units.

FIG. 3D illustrates a mass spectrum of bradykinin following treatment ofthe monocation of bradykinin with a hexacation of poly(propylenimine)(1,4-diaminobutane (DAB) core, generation 4) dendrimer showing only the[B+H]⁺ ion and none of the [B+2H]²⁺ ion. The vertical axis is relativeabundance in arbitrary units.

DETAILED DESCRIPTION OF THE INVENTION

The initial charge on a molecule or analyte to be ultimately analyzed bymass spectrometry may be generated by any of the variety of techniquesincluding but not limited to those commonly employed in massspectrometric analysis, such as plasma desorption ionization (PDI),field desorption ionization (FDI), electrohydrodynamic ionization (EHI),laser desorption ionization (LDI), matrix assisted laser desorptionionization (MALDI), atmospheric pressure matrix assisted laserdesorption ionization (APMALDI), spray ionization techniques, includingelectrospray ionization (ESI), nano-electrospray ionization, thermosprayionization (TSI), aerospray ionization (ASI), sonic spray ionization(SSI), particle desorption methods such as fast atom bombardment (FAB),fast ion bombardment (FIB), such as secondary ion mass spectrometry(SIMS), and massive cluster impact, and the like. The initial ionizingsource includes but is not limited to fast atoms such as xenon, argon,and the like, slow electrons, high energy electrons, and clusters suchas glycerol clusters, and the like. It is appreciated that the processesdescribed herein are not limited by the method used to generate the ionsused in these processes, and further that the ionization method may useeither charged or uncharged substances to generate the initial charge onthe molecule or analyte.

It is appreciated that depending upon the nature of the analyte ormolecule to be detected, either a positive charge or a negative chargemay be generated during initial ionization, and therefore the selectionof the ionization technique is conveniently chosen after considering thenature of the analyte or molecule. Further, in order for an analysis totake place, a variety of procedures may be followed to introduce asample containing the analyte or molecule into the apparatus wherein theinitial charge is generated. Those procedures include but are notlimited to those common to the field of mass spectrometry, such asintroduction of the sample as a solution, which is aspirated,volatilized, or atomized, or introduction of the sample as a solid orliquid, which may be adhered to a probe or other solid surface, such asa matrix.

It is also appreciated that certain molecules may form a positive chargemore easily than a negative charge, while other molecules may form anegative charge more easily than a positive charge. For example,ambident molecules, such as peptides and proteins, may form withrelative ease either a positive ion or a negative ion. However, certaincarbohydrates may form positive ions more readily than negative ions. Incontrast, certain oligonucleotides and nucleic acids may form negativecharges more readily than positive ions.

Peptides and proteins are analyzed by the processes and apparatusdescribed herein, and may be widely varying in both primary sequence andmolecular weight. Peptides and proteins, that may be analyzed includebut are not limited to bradykinin, ubiquitin, cytochrome C, hemoglobin,and the like. Oligosaccharides and carbohydrates are analyzed by theprocesses and apparatus described herein, and may be widely varying inboth primary sequence and molecular weight. The oligonucleotides andnucleic acids that may be analyzed by the processes described herein arewidely varying in both primary sequence and molecular weight. Theoligonucleotides and nucleic acids that may be analyzed include but arenot limited to oligonucleotides isolated from natural sources andsynthetic oligonucleotides, having illustratively from about 5 to about50 nucleotides. In addition, nucleic acids, including nucleic acids fromplants, animals, bacteria, and the like, including very large nucleicacids that have more than about 100 kilobases may be analyzed using theprocesses and apparatus described herein.

It is understood that any molecule that may be ionized into a gaseousstate using any of the techniques described herein or any othertechnique known to produce ionized molecules in the gas phase, may beanalyzed by the processes described herein provided that charge statesof both polarities may be formed on the molecule, and that at least oneof those charge states has an absolute value greater than one. Thoughthe generation of multiple charges on a molecule or analyte prior toanalysis is more often desired in molecules and analytes having highmolecular weights, the processes and apparatus described herein areequally applicable to the analysis of smaller molecules, where it isdesirable to generate higher charge states or multiple charges thereon.Without being bound by theory, the generation of multiple charges on amolecule may account for an increase in the number of fragmentsgenerated, and to the degree of, and the diversity of the fragmentationobserved. It is appreciated that increased fragmentation and increasedfragmentation diversity may enhance the ability to identify the chemicalcomposition or chemical structure, including the primary sequence oflarge molecules, such as various biological compounds includingproteins, nucleic acids, and polysaccharides.

Depending upon the technique used to generate the initial charge, anddepending upon the nature of the molecule to be detected, the initialcharge may therefore be positive or negative. In addition, the initialcharge on the molecule may have an absolute value of one oralternatively, the initial charge on the molecule may have an absolutevalue greater than one. In the case where the initial charge is greaterthan one, the initial charge may be optionally concentrated to a lowerselected charge by using a suitably configured concentrating componentthat uses the technique of ion parking. Ion parking is generallydescribed in U.S. Pat. No. 4,849,628 to McLuckey et al., the disclosureof which is incorporated herein by reference. It is appreciate that thetechnique of ion parking is advantageously used in processes where themolecular ions or fragment ions form a population of charge states ofsimultaneously varying values. Such a condition may complicate theinterpretation of the resulting mass spectrum. Concentrating the variouscharge states to a single state will generally simplify the massspectrum obtained.

Alternatively, using a conveniently placed component such as anatmospheric sampling glow discharge ionization (ASGDI) source, and thelike, the absolute value of the initial charge may be reduced one chargeat a time, in a stepwise fashion. An ASGDI is capable if reducing theabsolute value of both positively charged and negatively chargedmolecular ions or fragments thereof In one aspect the ASGDI source usesmonoanions of perfluorocarbons, such asperfluoro-1,3-dimethylcyclohexane (PDCH). In the case where the initialcharge is both greater than one and positive, the initial charge may beoptionally lowered to a lower selected charge, including a charge ofnegative one, by using a suitably configured electron capturedissociation (ECD) component.

In either the case of an initial ionization having an absolute value ofone or greater than one, the charge of the molecular ion so generated,may be changed to a charge having an absolute value equal to or greaterthan one, but of opposite polarity to the charge generated initially.Such a conversion is accomplished by contacting the molecular ion,illustratively in the gas phase, with a multiply charged ionic compound.A great variety of multiply charged ionic compounds may be used for thispurpose of polarity change in the processes described herein.

The multiply charged ionic compounds useful in the processes describedherein include but are not limited to polyionic compounds havingmultiple functional groups that may each form a net charge or aplurality of charges under conditions that may be created in typicalmass spectrometric analyses and apparatus. Such molecules include butare not limited to compounds having a plurality of amine groups,heteroaromatic amine groups, imine groups, hydrazine groups, guanidinegroups, carboxylic acid groups, sulfonic acid groups, phosphonic acidgroups, and the like. The multiply charged ionic compounds describedherein include polymeric materials, such as dendrimers, and naturallyoccurring polymeric materials such as nucleic acids and proteins.Illustratively, (5′-d(AAAA)-3′)³⁻, (melittin+4H)⁴⁺, (horsemyoglobin+20H)²⁰⁺, (holomyoglobin+9H)⁹⁺, and cytochrome C andapomyoglobin carrying multiple charges are useful in the processesdescribed herein.

It is appreciated that such compounds may possess a mixture oflike-charged groups, such as compounds that possess both carboxylic acidgroups and sulfonic acid groups, or compounds that possess both ammoniumand iminium groups. Further, it is appreciated that a mixture ofcompounds may be used in the processes described herein for changing thecharge on the analyte or molecule to be detected, such as a mixture of apolyamine compound and a polyimine compound.

Multiply charged ionic compounds having a plurality of negativelycharged groups include aromatic groups having a plurality of carboxylicacid groups present thereon, including alkylcarboxylates,cycloalkylcarboxylates, arylcarboxylates, such as phenyl-dicarboxylic,-tricarboxylic, and tetracarboxylic acids, arylalkylcarboxylates, suchas phenyl-dipropionic, tripropionic, and tetrapropionic acids, andphenyl-diacetic, triacetic, and tetraacetic acids, and the like. Inaddition, dendrimers having multiple carboxylic acid, sulfonic acid, orphosphonic acid functionalities are useful in the processes describedherein.

Multiply charged ionic compounds having a plurality of positivelycharged groups include aminoalkyl, hydrazinoalkyl, guanidinoalkyl,aminocycloalkyl, hydrazinocycloalkyl, guanidinocycloalkyl, aminoaryl,hydrazinoaryl, guanidinoaryl, heteroaromatic amines, such as pyridinyls,pyrimidinyls, pyridazinyls, pyrazinyls, imidazolinyls, oxazolihnyls,pyrrolyls, pyrazolyls, indolyls, quinolinyls, quinazolinyls groups, andthe like. Illustratively, dendrimers having multiple ammonium or iminiumgroups, including alkyleneammonium and alkyleneiminium functionalitiesare useful in the processes described herein. Dendrimers useful in theprocesses described herein include but are not limited tocyclotriphosphazene-phenoxymethyl(methylhydrazono) (PMMH) dendrimers,Polypropylenimine hexadecaamine Dendrimers, Polypropyleniminedotriacontaamine Dendrimers, Polypropylenimine tetraamine Dendrimers,Polypropylenimine tetrahexacontaamine Dendrimers, any of the variouspolyamidoamine (PAMAM) dendrimers, and any of the various primary-amineterminated dendrimers.

It is understood that many dendrimers are commercially available as amixture of dendrimers with a single compound having a specified numberof chargeable functional groups present thereon. In these cases, it isappreciated that following the preparation of the multiply charged ioniccompound from the dendrimer, a mass selection procedure may beadvantageously included as a step in the processes described herein.Such a mass selection procedure will have the benefit of decreasing thenoise observed in the final mass spectrum and simplifying theinterpretation of the resulting mass spectrum. The mass selectionprocess may be accomplished using any of a variety of techniques commonto mass spectrometry, including quadrupole ion traps, linear ion traps,time of flight (TOF), and ion cyclotron resonance. In such a step, thelower and higher molecular weight compounds, as well as the compoundsthat do not have the desired number of chargeable groups present in themultiply charged ionic compound precursor are removed. The resultingpurified multiply charged ionic compound may then be contacted with themolecule or analyte. Similarly, in certain aspects of the processesdescribed herein, a mass selection or mass filtering step may beincluded where the initially generated molecular ions or fragment ionsthereof are separated. In these embodiments, only certain chargedionization states, or certain mass ranges are contacted with themultiply charged ionic compounds. A process that uses the principle oftandem mass spectrometry may include such a filtering or selectionprocedure.

It is also appreciated that during the course of a polarity change onthe molecule or fragment thereof, in some embodiments of the invention,aggregates of these ions and the multiply charged ionic compound mayform. These aggregates also have the overall effect of changing thecharge on the molecule, including changing the charge on the molecule toa value opposite in polarity. However, in these cases the charge ischanged by the aggregation of the multiply charged ionic compound withthe analyte ion rather than the transfer of either an electron or aproton between the multiply charged ionic compound and the analyte ormolecular ion. In either case, the detection of the analyte by massspectrometry is accomplished by evaluating the masses that are producedin the apparatus. Mustratively, a protein having an initial charge offor example of positive one, may form an aggregate in the ion trap witha multiply charged ionic compound that is for example anaryl(tetraalkylenecarboxylate), where the aggregate has an overallcharge of negative three.

In configurations of the processes described herein where there isincluded a tandem mass spectrometry procedure, also known as MS/MS, itis understood that such aggregates may yet be analyzed by thesetechniques commonly in use, following a heat treating step, where theaggregate molecule comprising the analyte or molecular ion and themultiply charged ionic compound are separated in a fashion where theanalyte of molecular ion retains the increased charge state.

The polarity change of the initial charge on the analyte or molecule orfragment thereof may result in a second charge state that has anabsolute value greater than or equal to the absolute value of theinitial charge state. In other aspects, the polarity change of theinitial charge on the analyte or molecule or fragment thereof may resultin a second charge state that has an absolute value less than or equalto the absolute value of the initial charge state. Further, the thirdcharge may have an absolute value greater than or equal to the absolutevalue of the second charge state. Illustratively, the third charge statehas an absolute value greater than the absolute value of the initialcharge on the analyte or molecule or fragment thereof.

It is understood that the molecule or analyte may undergo fragmentationat various times during the operation of the processes described hereinto produce fragment ions, and at various times during the modificationof the charges thereon. The foregoing discussion regarding thesubsequent processes and process steps apply equally to these fragmentions as to the molecular ions or analyte ions described above. Forexample, a protein-based analyte may illustratively fragment uponinitial ionization to a plurality of charged ion fragments, each havinga first charge that may be the same or different. The unfragmentedmolecular ion may also be included in this group. Each molecular ion orfragment ion thereof is then contacted with a multiply charged ioniccompound, or a mixture of such compounds that change the charge state toa second charged state, the polarity of which is the opposite of theinitial charge state. Optionally, this group of now-oppositely chargedmolecular or molecular fragment ions may be contacted with a secondmultiply charged ionic compound, or a second mixture of such compoundscapable of changing the charge state to a third charge, where the thirdcharge on any given molecular or molecular fragment ion is the same ordifferent, but where each is the opposite polarity of the second charge.Additional fragmentation of the molecular ion or other fragmentationions produced in the initial ionization may occur following either orboth steps wherein the molecular ions or fragment ions are contactedwith the one of more multiply charged ionic compounds. In the case ofmultiply charged molecular ions or fragment ions, further ordifferential fragmentation is optionally accomplished by including anelectron capture dissociation (ECD) procedure, a collision-induceddissociation procedure, and the like.

As described herein, a mass filtering or mass selection procedure mayalso be introduced in the process following the modification of theinitial or even the second charge state on the molecule or fragmentthereof. This mass filtering or mass selection procedure is optionallyaccomplished using a scanning technique, such as by scanning the massesfrom a linear or quadrupole ion trap, and the like.

It is appreciated that the change between the first and second chargestates, and the change between the second and third charge states mayoccur as a stepwise procedure, wherein a dwell time is introduced intothe process to allow a substantial number of molecules or molecularfragments thereof to change charge states. Subsequently, the changebetween the second and third charge states is performed.

Alternatively, the change among charge states may occur in acontemporaneous manner, wherein a mixture of multiply charged ionicreagents or compounds comprises both positively charged ionic compoundsand negatively charged ionic compounds. In this variation of theprocess, the molecular ion or molecular fragment ion undergoes twochanges in polarity, each oppositely directed, in a one-step process. Itis appreciated that the selection of positive and negative multiplycharged ionic compounds is accomplished in a way to enhance thepopulation of molecular ions or fragment ions that have a desired chargestate. This enhancement may be derived through an understanding of thethermodynamics controlling the overall modification of the charge stateon the molecular ion or fragment ion thereof. In one aspect, desirablethermodynamics favor the formation of a charge state on the molecularion or molecular fragment ion that is the same as the initial chargestate thereon, passing through a second charge state that is theopposite of the initial charge state, and having a final charge with anabsolute value that is greater than the absolute value of the initialcharge state. In one illustrative aspect, the second charge state has anabsolute value of one. In another illustrative embodiment, both theinitial charge state and the second charge state have an absolute valueof one, and the third charge state has an absolute value greater thanone.

The molecular ions generated in the processes described herein may alsobe analyzed, where such analysis includes detection of molecular ions orfragments thereof, and measurement of the masses of these molecular ionsor fragments thereof that are produced in the processes or by theapparatus described herein. Such detection and measurement may beperformed by using any of the variety of techniques commonly employed inmass spectrometric analysis, including but not limited to scanning fromquadrupole ion traps, linear ion traps, analysis via time of flight(TOF), ion cyclotron resonance, and the like. Further, the detection andanalysis may also include a tandem mass spectrometry configuration.

The processes described herein may be performed by any variety of massspectrometry equipment that is common to mass spectrometric analysis andhas been configured with devices or components that are capable ofperforming the procedures required by the processes described herein. Ingeneral, the apparatus described herein are constructed from componentsor devices that are capable of performing functions required by theprocesses described herein, such as (1) various sources for generatingions or charges on molecules or analytes to be analyzed, (2) variouscomponents for storing or trapping those molecular ions or fragment ionsthereof so-generated, including components for directing ions to suchstorage components, (3) various components for optionally filtering orselecting certain masses from those molecular ions or fragment ionsthereof, (4) various components for providing a reaction environment forcontacting other charged or multiply charged ionic compounds with thosemolecular ions or fragment ions thereof to modify the charge states ofthe molecular ions or fragment ions, and (5) various components foranalyzing the molecular ions or fragment ions thereof. It is appreciatedthat the apparatus described herein may also be capable of performingother processes in addition to those described herein. Such flexibilitypossessed by an apparatus should not be understood to suggest thatapparatus is excluded from the invention described herein

FIG. 1 illustrates one such configuration 10. This configuration isintended to illustrate one of many possible configurations of apparatuscapable of performing the processes described herein. The configurationcomprises a first housing component 12 and a second housing component14. The first housing component 12 possesses a first generating source16, a second generating source 18, and a third generating source 20. Thegenerating sources 16, 18, and 20 each interface with the first housingcomponent 12 through first, second, and third interface regions 22, 24,and 26, respectively, such as a first, second, and third atmospherevacuum interface. The interface regions 22, 24, and 26 are each coupledto a first, second, and third general ion optical element 28, 30, and32, respectively, such as a first, second, and third electrostatic lensassembly, each comprising lenses L1, L2, and L3. Each general ionoptical element 28, 30, and 32 is aimed into a turning quadrupole 34which is capable of diverting the ions generated from the generatingsources 16, 18, and 20 into an ion transmission device 36, such as atube lens or a quadrupole mass selector. The ion transmission device 36couples the first housing component 12 to the second housing component14. In addition, the ion transmission device 36 is coupled to a fifthgeneral ion optical element 38, which is aimed into a quadrupole iontrap 46.

The second housing component 14 possesses a fourth generating source 40,such as an atmospheric sampling glow discharge ionization source,interfaced with the second housing component 14 through a fourthinterface region 42. The fourth interface region 42 is coupled to afourth general ion optical element 44 which is aimed into the quadrupoleion trap 46. The charges on ions that have been generated in the firsthousing component 12 and diverted into the ion trap 46 in the secondhousing component may be changed by the introduction of multiply chargedionic compounds as described herein, and generated illustratively in thefirst housing component 12, or by ions generated from the fourth ionsource 40. The second housing component 14 also possesses a detector 48,such as an electron multiplier, for detecting ions released or ejectedfrom the ion trap 46.

In FIG. 1, the apparatus is configured such that multiple sources arepresent for the optional generation of the various positively andnegatively charged molecules, analytes, or multiply charged ioniccompounds described herein. It is appreciated that all of these sourcesmay not be required in some variations of the process. Further, thesources may be of the same character, such as a plurality of ESIsources, or of a different character, such as an ESI, a LSI, and a SIMSsource. Any combination of sources occupying the various bays may beincluded in the apparatus described herein. Illustratively, in oneembodiment, the three sources may be an ESI source, a LDI source, and aFAB(+) source; while in another illustrative embodiment, the threesources may be a negative ESI source, a positive ESI source, and anAPMALDI source.

In addition, FIG. 1 illustrates an apparatus where there are twocomponents, the first comprising the generating sources and a tuningquadrupole, and the second component comprising the storage andcontacting area and detecting features. It is appreciated that in otherembodiments, all of these functional pieces may be configured in thesame component, while in other illustrative embodiments, an additionalcomponent may house, for example, the detecting feature separate fromthe contacting area.

Illustratively, one source is used to generate an initial charge on themolecular ion. As described herein, the generation of the molecular ionmay be accomplished using any of a variety of components that arecapable of generating ions using plasma desorption ionization (PDI),field desorption ionization (FDI), electrohydrodynamic ionization (EHI),laser desorption ionization (LDI), matrix assisted laser desorptionionization (MALDI), atmospheric pressure matrix assisted laserdesorption ionization (APMALDI), spray ionization techniques, includingelectrospray ionization (ESI), nano-electrospray ionization, thermosprayionization (TSI), aerospray ionization (ASI), sonic spray ionization(SSI), particle desorption methods such as fast atom bombardment (FAB),fast ion bombardment (FIB), such as secondary ion mass spectrometry(SIMS), and massive cluster impact, and the like.

In one embodiment, the source is a SSI source capable of generating bothpositively and negatively charged multiply charged ionic compounds. Inthat embodiment, the molecular ion or fragment ion may be sequentiallycontacted with each of these ionic compounds generated from the samesource. In one illustrative configuration, the SSI source is combinedwith a linear ion trap. This configuration of the apparatus describedherein is well-suited for a flow through process, where the sourcegenerates the initial charge on the molecule, and the resulting ions orfragment ions thereof are placed in the storage trap. Subsequently, theSSI source is used to generate a multiply charged ionic compound orcompounds having one polarity. These ions are allowed to flow throughthe linear ion trap, which unlike a quadrupole ion trap is capable ofholding only one polarity of charge at a time. After some predeterminedtime, the SSI source is used to generate a multiply charged ioniccompound or compounds having the other polarity. This ion stream is thencontacted with the molecular ions or fragment ions in the linear trap.

The apparatus described herein is illustratively configured to include aturning quadrupole capable of directing the ions so produced into astorage device or trap, such as a quadrupole ion trap or a linear iontrap. In order that multiple sources may be conveniently placed on theapparatus, a turning quadrupole may advantageously be used to direct theions of interest into other components of the apparatus, such as thecontacting or reaction vessel component. The various ions generated fromthe various corresponding sources are directed into the turningquadrupole by an electrostatic lens, a series of electrostatic lenses,an electrostatic lens assembly, such as an Einzellens, or other suitablyconfigured general ion optical element. Initially, the molecular ion orfragment thereof is turned into the contacting component or storagetrap.

In an alternate embodiment, the generating component may be configuredto include a mass selecting component or mass filtering component, suchas a quadrupole ion mass filter, linear ion mass filter, and the like.The mass selecting component will have the effect of purifying the ions,such as the molecular ions or the fragment ions, that are introducedinto the contacting component via the turning quadrupole. Thispurification procedure may be applied to the initially generatedmolecular ions, or to any of the multiply charged ionic compounds usedto modify the charges on the molecular ions or fragment ions thereofduring the contacting procedure. Each source may be optionallyconfigured with such a mass selecting component.

Apparatus described herein also include a contacting or reaction vesselcomponent. In some embodiments, the contacting or reaction vesselcomponent is a quadrupole ion trap or a linear ion trap. The contactingor reaction vessel component allows for the molecular ions or fragmentions initially generated, and optionally filtered to be contacted withmultiply charged ionic compounds that will change the charges on themolecular ions or fragment ions. It is appreciated, that like themolecular ions or fragment ions that are initially generated, thecharges generated on the multiply charged ionic compounds may also bemass filtered or mass selected prior to introduction into the reactionvessel component.

The contacting component includes a trap for the purpose of holding fora predetermined period of time the molecular ions or fragments ionsthereof in contact with any one of the multiply charged ionic compounds.In addition, the trap portion of the contacting component has thepurpose of holding the molecular ions or fragments ions thereof forsubsequent ion concentration or ion parking procedures. In someconfigurations of the apparatus described herein, the ion storage deviceor ion trap is a linear ion trap or a quadrupole ion trap. A linear iontrap is a two-dimensional trap that may only store one charge statepolarity at a time. A quadrupole ion trap is a three-dimensional trapthat may store two charge state polarities at a time.

In apparatus where the generating component and the contacting componentare separate, the generating component and the contacting component maybe in fluid communication by a suitably placed ion transmission system,such as an electrostatic filter assembly, tube lens, an RF ion guide,and the like, or an electrodynamic ion transmission system such as anhexapole or an octapole, and the like. Alternatively, the tube lens maybe configured to include a single mass selecting component that can beused as a mass filter of any substance generated by the various ionizingsources and directed into the ion trap. In this embodiment, thenecessity of having a mass selecting component at each source may bethus alleviated.

Further, the contacting component or reaction chamber may also include amass selecting or mass filtering component, a mass scanning component,and a mass detecting component, such as an electron multiplier. It is tobe understood that while the apparatus illustrated in FIG. 1 uses acontacting component that includes a trap, a mass selecting or massfiltering component, and a mass scanning component embodied in aquadrupole ion trap, these components may be configured as separatecomponents in alternative embodiments of the apparatus described herein.

Illustratively, the mass scanning component may be a TOF componentrather than a quadrupole mass filter. In that configuration, aquadrupole ion trap is used to store and contact the molecular ions, andthe TOF component is used to analyze the masses resulting from thecharge modification. In addition, the mass storage and contactingcomponent may be a linear ion trap, where the multiply charged ioniccompounds are contacted with the molecular ion or fragment ion in aflow-type process rather than the batch-type process illustrated by thequadrupole ion storage embodiment. In this configuration, a SSI sourceis conveniently selected as it has the ability to generate both positiveand negative ions, and is particularly suited for this type offlow-through embodiment. However, it is appreciated that variousconfigurations of these components may be constructed to perform theprocesses described herein.

In some configurations of the apparatus described herein, analyte ionsor fragment ions thereof, or molecular ions or fragment ions thereof aredetected. Detection is accomplished by any of the variety of techniquescommon to the field of mass spectrometry, and includes electronmultipliers, and the like. Prior to the detection, a mass selection ormass filtering procedure is optionally performed on the analyte ions orfragment ions thereof or molecular ions or fragment ions thereof. Such amass filtering or mass selection procedure may be accomplished byscanning the masses with a quadrupole. In other embodiments, the massfiltering or mass selection procedure may be accomplished using a timeof flight component that in one step permits separation of the masses.

In embodiments having a quadrupole ion trap serving as the contactingcomponent, the ions to be analyzed and detected are released using anyof a variety of common techniques, such as resonance ejection and thelike, into the detecting component. In one aspect the detectingcomponent is an electron multiplier and the like. In other aspects, thedetecting component includes a conversion dynode.

The foregoing discussion sets forth many possible embodiments withvarious aspects of the invention described herein for both the processesand apparatus. However, it is appreciated that other embodiments arisingfrom combinations of the components described herein or the proceduresdescribed herein though not explicitly set forth are neverthelesscontemplated to fall within the scope of the invention as describedherein. Further, the following illustrative examples are presented toprovide an additional understanding of the nature and the spirit of theinvention. The examples are intended to be illustrative and should notbe considered to limit the invention in any way.

EXAMPLE 1 Mass Spectrometric Analysis of Bradykinin with PDPA andPoly(propyleneimine) Dendrimer

FIG. 2 summarizes results for an experiment in which bradykinin wassubjected to two steps of charge inversion. FIG. 2A illustrates thepositive ion electrospray mass spectrum of bradykinin after singlypredominated ions of the protein were isolated in a quadrupole ion trap.FIG. 2B shows the spectrum that resulted after the bradykinin (M+H)⁺ ionwas contacted with multiply charged ionic compound para-dibenzopropanoicacid (PDPA−2H)²⁻. The formation of singly deprotonated bradykinin (M−H)⁻is observed in the spectrum shown in FIG. 2B. FIG. 2C shows the positiveion spectrum that resulted from the reaction of the multiply chargedionic compound (poly(propyleneimine) dendrimer+6H)⁶⁺. The bradykinin(B+H)⁺ ion reappears in addition to the bradykinin (B+2H)²⁺ cation. Amass selection step was not performed on the multiply charged dendrimer.Therefore, the presence of various charge states of that dendrimer areseen as low level signals in the resulting mass spectrum shown in FIG.2C.

EXAMPLE 2 Mass Spectrometric Analysis of Bradykinin with DAB Dendrimerand PAMAM Dendrimer

FIG. 3 summarizes results for an experiment in which two steps of chargeinversion were used to form the (B+2H)²⁺ ions from the (B+H)⁺ ions ofbradykinin. The overall conversion of bradykinin to the (B+2H)²⁺ ion isillustrated in FIG. 4. Solutions for nano-electrospray were prepared atconcentrations of 1 mg/mL bradykinin and 5 mg/mL DAB dendrimers inaqueous 1% and 5% acetic acid (positive ions) or 1 mg/mL PAMAM dendrimerin 2% NH₄HCO₃ (negative ions). A full description of the ion trap massspectrometer equipped with three ESI sources and ion optics that allowsequential injection of opposite polarity ions has been described inBadman et al., Anal. Chem., 74, 6237-43 (2002), the disclosure of whichis incorporated herein by reference. The first step of the experimentinvolved the accumulation of bradykinin ions formed via positiveelectrospray ionization in a quadrupole ion trap followed by isolationof the bradykinin (B+H)⁺ ion. The resulting mass spectrum is shown inFIG. 3A. A population of anions formed via negative electrosprayionization of a carboxylate terminated polyamidoamine dendrimer(generation 0.5) (PAMAM) was then admitted into the ion trap and allowedto react with the bradykinin (B+H)⁺ ions. The electrospray mass spectrumof the PAMAM reagent anions (no cations present) is shown in FIG. 3B.After reaction with the bradykinin (B+H)⁺ ions, both residual PAMAManions and (B−H)⁻ bradykinin anions were observed in the spectrum (datanot shown). The bradykinin (B−H)⁻ ions were then isolated and apopulation of poly(propylenimine) (1,4-diaminobutane (DAB) core,generation 4)dendrimer cations formed via positive ion electrospray wasadmitted into the ion trap and allowed to react with the anions. Theresulting product ion spectrum is shown in FIG. 3C. For comparison, thespectrum resulting from the sequence used to acquire the spectrum ofFIG. 3C but without the admission of anions is shown in FIG. 3D. Thelatter spectrum shows the mixture of bradykinin (B+H)⁺ and DAB dendrimercations that resulted from the sequential injection of positive ionswithout the intervening anion accumulation and ion/ion reaction period.The position of the (B+2H)²⁺ bradykinin ion on the mass-to-charge scale,if it were present, is indicated by an arrow. Several major differencescan be noted in the comparison of FIGS. 3C and 3D. First, the chargestate distribution of the DAB dendrimer ions has shifted to lower chargestates as a result of partial neutralization with the bradykinin anions.Second, the abundance of the (B+H)⁺ ion is significantly reduced (byroughly a factor of ten) by the admission of the anions. This ispresumably due to losses associated with neutralization of part of the(B+H)⁺ ion population by the PAMAM anions used to form (B−H)⁻ ions,losses associated with neutralization of the (B−H)⁻ anions in reactionswith DAB cations, and losses associated with the fraction of ions formedas (B+2H)²⁺ ions. Third, there is a clear signal that arises from theformation of the bradykinin (B+2H)²⁺ ion. A roughly 200-fold increaseover the background signal at the m/z ratio of the (B+2H)²⁺ ion in FIG.3D is observed in FIG. 3C. Net yields for bradykinin in the net reactionof (B+H)⁺→(B+H)⁻→(B+2H)²⁺ have been observed as high a 20% dependingupon the reaction time permitted.

1. A process for analyzing a molecule by mass spectrometry, comprising:(a) generating a charge on the molecule; and (b) changing the charge onthe molecule to a different value and having the opposite polarity. 2.The process of claim 1, wherein the changing step includes changing thecharge to a number, where the absolute value of the number is greaterthan the absolute value of the charge.
 3. The process of claim 1,wherein the changing step includes changing the charge to a number,where the charge has an absolute value greater than one, and the numberhas an absolute value of one.
 4. A process for analyzing a molecule bymass spectrometry, comprising: (a) generating a first charge on themolecule; (b) changing the first charge on the molecule to a secondcharge; and (c) changing the second charge on the molecule to a thirdcharge; where the second charge has the opposite polarity of the firstcharge, or the third charge has the opposite polarity of the secondcharge, or where the second charge has the opposite polarity of thefirst charge, and the third charge has the opposite polarity of thesecond charge.
 5. The process of claim 4, wherein the generating stepincludes generating a first charge on the molecule where the molecule isselected from the group consisting of proteins, peptides, nucleic acids,oligonucleotides, oligosaccharides, and carbohydrates.
 6. The process ofclaim 4, wherein the generating step includes generating a first chargeon the molecule using a technique selected from the group consisting ofplasma desorption ionization, field desorption ionization,electrohydrodynamic ionization, laser desorption ionization, matrixassisted laser desorption ionization, atmospheric pressure matrixassisted laser desorption ionization, electrospray ionization,nano-electrospray ionization, thermospray ionization, aerosprayionization, sonic spray ionization, fast atom bombardment, fast ionbombardment, secondary ion mass spectrometry, and massive clusterimpact.
 7. The process of claim 4, wherein the changing step includeschanging the first charge to a second charge, where the absolute valueof the second charge is equal to or less than the absolute value of thefirst charge.
 8. The process of claim 4, wherein the changing stepincludes changing the first charge to a second charge, where theabsolute value of the second charge is less than the absolute value ofthe first charge.
 9. The process of claim 4, wherein the changing stepincludes changing the first charge to a second charge by contacting themolecule with a multiply charged ionic compound having a plurality ofcharged groups.
 10. The process of claim 9, wherein the changing stepincludes changing the first charge to the second charge, where the firstcharge is a negative charge, by contacting the molecule with a multiplycharged ionic compound having a plurality of charged groups selectedfrom the group consisting of ammonium groups, iminium groups, andprotonated heteroaromatic groups.
 11. The process of claim 10, whereinthe changing step includes changing the first charge to a second chargeby contacting the molecule with a multiply charged dendrimer.
 12. Theprocess of claim 9, wherein the changing step includes changing thefirst charge to a second charge, where the first charge is a positivecharge, by contacting the molecule with a multiply charged ioniccompound having a plurality of charged groups selected from the groupconsisting of hydroxide groups, carboxylate groups, sulfonate groups,and phosphonate groups.
 13. The process of claim 12, wherein thechanging step includes changing the first charge to a second charge bycontacting the molecule with a multiply charged dendrimer.
 14. Theprocess of claim 4, wherein the changing step includes changing thefirst charge to the second charge, where the molecule is in the gasphase.
 15. The process of claim 4, wherein the changing step includeschanging the first charge to a second charge in an ion storage trapselected from the group consisting of quadrupole ion traps and linearion traps.
 16. The process of claim 4, wherein the changing stepincludes changing the second charge to a third charge, where theabsolute value of the third charge is equal to or greater than theabsolute value of the second charge.
 17. The process of claim 4, whereinthe changing step includes changing the second charge to a third charge,where the absolute value of the third charge is greater than theabsolute value of the second charge.
 18. The process of claim 4, whereinthe changing step includes changing the second charge to a third chargeby contacting the molecule with a multiply charged ionic compound havinga plurality of charged groups.
 19. The process of claim 18, wherein thechanging step includes changing the first charge to the second charge,where the first charge is a negative charge, by contacting the moleculewith a multiply charged ionic compound having a plurality of chargedgroups selected from the group consisting of ammonium groups, iminiumgroups, and protonated heteroaromatic groups.
 20. The process of claim19, wherein the changing step includes changing the first charge to asecond charge by contacting the molecule with a multiply chargeddendrimer.
 21. The process of claim 18, wherein the changing stepincludes changing the first charge to a second charge, where the firstcharge is a positive charge, by contacting the molecule with a multiplycharged ionic compound having a plurality of charged groups selectedfrom the group consisting of hydroxide groups, carboxylate groups,sulfonate groups, and phosphonate groups.
 22. The process of claim 21,wherein the changing step includes changing the first charge to a secondcharge by contacting the molecule with a multiply charged dendrimer. 23.The process of claim 4, wherein the changing step includes changing thefirst charge to the second charge, where the molecule is in the gasphase.
 24. The process of claim 4, wherein the changing step includeschanging the first charge to a second charge in an ion storage trapselected from the group consisting of quadrupole ion traps and linearion traps.
 25. The process of claim 4, wherein the changing stepincludes changing the second charge to a third charge in an ion storagetrap selected from the group consisting of quadrupole ion traps andlinear ion traps.
 26. The process of claim 4, wherein the changing stepincludes changing the second charge to a third charge, where the secondcharge is the opposite polarity of the third charge.
 27. The process ofclaim 26, wherein the changing step includes changing the second chargeto a third charge, where the second charge is a negative charge, bycontacting the molecule with a compound selected from the groupconsisting of poly(alkyleneinine) dendrimers and poly(alkyleneamine)dendrimers.
 28. The process of claim 27, wherein the changing stepincludes changing the second charge to the third charge, where thesecond charge is a positive charge, by contacting the molecule with acompound selected from the group consisting of poly(alkylenecarboxylicacid) dendrimers and poly(alkylenesulfonic acid) dendrimers.
 29. Theprocess of claim 4, further comprising the step of concentrating thethird charge using ion parking.
 30. The process of claim 4, furthercomprising detecting the molecule or a fragment thereof using tandemmass spectrometry.
 31. The process of claim 30, further comprisingconcentrating the charge states of the molecule or a fragment thereofprior to the detecting step.
 32. The process of claim 4, furthercomprising lowering the absolute value of the third charge by at leastone using an atmospheric sampling glow discharge ionization source. 33.The process of claim 32, wherein the lowering step is performed prior tothe detecting step.
 34. The process of claim 4, further comprisingidentifying the molecule or a fragment thereof.
 35. A process foranalyzing a molecule by mass spectrometry, comprising: (a) generating afirst charge on the molecule; (b) changing the first charge on themolecule to a second charge having the opposite polarity of the firstcharge; and (c) changing the second charge on the molecule to a thirdcharge having the opposite polarity of the second charge.
 36. Theprocess of claim 35, wherein the step of changing the first charge andthe step of changing the second charge are performed contemporaneously,by contacting the molecule having the first charge, or a fragmentthereof, with a first multiply charged ionic compound, and a secondmultiply charged ionic compound, where the first ionic compound and thesecond ionic compound have charges of opposite polarity.
 37. A processfor analyzing a molecule by mass spectrometry, comprising generating afirst charge on the molecule having an absolute value equal to orgreater than one; changing the first charge on the molecule to a secondcharge having an absolute value of one, by contacting the molecule witha first multiply charged ionic compound; and changing the second chargeon the molecule to a third charge having an absolute value greater thanone, by contacting the molecule with a second multiply charged ioniccompound.
 38. The process of claim 37, wherein the generating stepincludes generating the first charge by plasma desorption ionization,field desorption ionization, electrohydrodynamic ionization, laserdesorption ionization, matrix assisted laser desorption ionization,atmospheric pressure matrix assisted laser desorption ionization,electrospray ionization, nano-electrospray ionization, thermosprayionization, aerospray ionization, sonic spray ionization, fast atombombardment, fast ion bombardment, secondary ion mass spectrometry, ormassive cluster impact.
 39. An apparatus for analyzing a molecule bymass spectrometry, comprising: (a) a first generating source capable ofionizing the molecule to a first charge; (b) a second generating sourcecapable of preparing a first multiply charged ionic compound; (c) athird generating source capable of preparing a second multiply chargedcompound capable of changing the second charge on the molecule to athird charge; and (d) an ion trap; where the first generating source,the second generating source, the third generating source, and the iontrap are in fluid communication.
 40. The apparatus of claim 39, whereinthe first generating source, the second generating source, and the thirdgenerating source comprise a first component; and the ion trap comprisesa second component.
 41. The apparatus of claim 40, wherein the firstcomponent is coupled to the second component with a tube lens, an radiofrequency ion guide, an hexapole, or an octapole.
 42. The apparatus ofclaim 40, further comprising a quadrupole mass filter, where the firstcomponent is coupled to the second component through the quadrupole massfilter.
 43. The apparatus of claim 40, wherein the second componentfurther comprises a concentrating source coupled to the ion trap. 44.The apparatus of claim 40, wherein second component further comprises anatmospheric sampling glow discharge ionization source.
 45. The apparatusof claim 39, further comprising a quadrupole mass filter, where at leastone of the group consisting of the first generating source, the secondgenerating source, and the third generating source includes thequadrupole mass filter.